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<p>The present study is the first attempt to standardize a genetic transformation protocol for <i>Nerium oleander</i> L. Initially, seedlings derived from zygotic embryos were utilized to optimize in vitro regeneration. Subsequently, nodal segments excised from in vitro–grown plantlets were employed to standardize multiple shoot induction on full-strength Schenk and Hildebrandt modified (FSHM) medium supplemented with four cytokinins—zeatin, 6-benzylaminopurine (BAP), thidiazuron (TDZ) and 6-furfurylaminopurine (FAP)—each at concentrations of 1, 2 and 4&#xa0;mg/L. Among these, zeatin proved most effective for explant survival and shoot induction across all tested concentrations, with 1&#xa0;mg/L yielding the highest mean number of shoots per explant (5.34 ± 0.07). The regenerated shoots were transferred to half-strength Murashige and Skoog (HMS) medium for rooting. The hygromycin sensitivity test indicated that 7&#xa0;mg/L concentration of hygromycin was optimal for the effective selection of transformants. For genetic transformation, <i>Agrobacterium tumefaciens</i> strain GV3101 harbouring the binary vector pCAMBIA1302, containing the monomeric green fluorescent protein (<i>mgfp</i>) and hygromycin resistance (<i>hptII</i>) genes, was employed. Nodal segments from in vitro plantlets and hypocotyls from 10-day-old seedlings were infected with the transformed <i>A. tumefaciens</i> at an optical density of 0.5 for 15, 30, and 45&#xa0;min, followed by co-cultivation in the dark for 72&#xa0;h. Thereafter, explants were transferred to shoot induction and selection media. Transformation was confirmed through PCR amplification of <i>hptII</i> and <i>mgfp</i> genes using both genomic DNA and cDNA. Fluorescence analysis further validated transgene expression. PCR results indicated that increasing infection duration reduced transformation efficiency in nodal segments, whereas it increased when the hypocotyl was used as an explant.</p>

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Standardization of in vitro regeneration and genetic transformation protocol in Nerium oleander L.

  • 期刊论文

  • 发表日期：

    2026年5月26日

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  • Sandhya Prajapati,
  • Renu Nimoriya,
  • Rosani Kumari Shaw,
  • Carol Janis Bilung,
  • Anjum Bano,
  • Dipak Kumar Mishra,
  • Vineeta Tripathi

摘要

The present study is the first attempt to standardize a genetic transformation protocol for Nerium oleander L. Initially, seedlings derived from zygotic embryos were utilized to optimize in vitro regeneration. Subsequently, nodal segments excised from in vitro–grown plantlets were employed to standardize multiple shoot induction on full-strength Schenk and Hildebrandt modified (FSHM) medium supplemented with four cytokinins—zeatin, 6-benzylaminopurine (BAP), thidiazuron (TDZ) and 6-furfurylaminopurine (FAP)—each at concentrations of 1, 2 and 4 mg/L. Among these, zeatin proved most effective for explant survival and shoot induction across all tested concentrations, with 1 mg/L yielding the highest mean number of shoots per explant (5.34 ± 0.07). The regenerated shoots were transferred to half-strength Murashige and Skoog (HMS) medium for rooting. The hygromycin sensitivity test indicated that 7 mg/L concentration of hygromycin was optimal for the effective selection of transformants. For genetic transformation, Agrobacterium tumefaciens strain GV3101 harbouring the binary vector pCAMBIA1302, containing the monomeric green fluorescent protein (mgfp) and hygromycin resistance (hptII) genes, was employed. Nodal segments from in vitro plantlets and hypocotyls from 10-day-old seedlings were infected with the transformed A. tumefaciens at an optical density of 0.5 for 15, 30, and 45 min, followed by co-cultivation in the dark for 72 h. Thereafter, explants were transferred to shoot induction and selection media. Transformation was confirmed through PCR amplification of hptII and mgfp genes using both genomic DNA and cDNA. Fluorescence analysis further validated transgene expression. PCR results indicated that increasing infection duration reduced transformation efficiency in nodal segments, whereas it increased when the hypocotyl was used as an explant.